Dimethylmaleimide: a new reagent for protein bioconjugation

Abstract

The amino acid cysteine has long been an attractive target for protein bioconjugation due to its rarity and unique reactivity among the nucleophilic side chains. Although the most widely used cysteine bioconjugation transformation involves maleimide, this system suffers from selectivity and reversibility issues as well as limited conjugation conditions. While investigating alternatives to this strategy, we discovered that dimethylmaleimide (DMM) formed stable, cysteine-specific conjugates in conditions where maleimide demonstrated nonspecific reactivity on a single cysteine variant of dihydrofolate reductase (DHFR). Furthermore, DMM analogs are synthetically accessible and can be attached to a variety of useful probes including fluorescent dyes and biotin tags. Using a solid-phase peptide screening approach, we discovered that DMM reactivity can be tuned using the amino acids surrounding the reactive thiol, achieving sitespecific conjugations to cysteine, lysine, and arginine residues. DMM poses an exciting and promising alternative to maleimide in a variety of areas, from creating probes for structural biology and proteomics to the production of biologics such as antibody-drug conjugates for cancer treatment. Support or Funding Information NIH Grant 5 T32 CA 9054-37Allergan, PLCLa Verne Noyes Fellowship Graphical abstract: Cysteine-selective bioconjugation of DHFR with dimethylmaleimide. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.